During lymphocyte development, antigen receptor genes are assembled from germline V,D and J gene segments by a site-specific somatic recombination process termed as V(D)J recombination. Defects in this pathway result in a number of primary immunodeficiencies like Severe Combined Immuno Deficiency (SCID) and Omenn syndrome and potentially can lead to development of leukemias and lymphomas. Two lymphoid specific proteins RAG1 and RAG2 cleave DNA between the coding segments and signal sequences. The Non-Homologous End Joining (NHEJ) DNA repair pathway repairs the double strand breaks thus generated. This reaction results in coding ends with covalently closed hairpin structures that need to be opened and processed before ligation. Mutations in a novel protein called Artemis causes RS-SCID syndrome, a SCID phenotype associated with radiosensitivity. Data from in vitro analysis and knockout mice have shown that Artemis is the nuclease that opens hairpin-coding ends. However, nothing is known about the biochemistry and structure of this protein. Further, direct evidence for its role in NHEJ is still lacking. Also, the C-terminal region of Artemis that forms almost half of the protein has not been studied. In this grant application, we describe a series of experiments that will address these issues while extending our observation that Artemis interacts with Ligase IV/XRCC4 in vivo through its C-terminal domain and this complex is very active in DSB ligation in vitro. Through specific aims proposed we will 1) characterize the biochemical properties of Artemis and develop its crystal structure, 2) investigate the in vivo relevance of the interaction of Artemis with Ligase IV in NHEJ and define a role of its C-terminal region in NHEJ and 3) establish the significance of this interaction in V(D)J recombination and ascertain the role of its C-terminal region. Together, the experiments proposed for the specific aims will begin to define the structure-function relationship of Artemis and the mechanism of different RS-SCID mutations found in humans. Further, by establishing the importance of interaction of Artemis with Ligase IV in both V(D)J recombination and NHEJ in vivo, we will provide novel insights into multiple roles played by Artemis in these pathways. Through this study, we will also describe a novel protein structure, by solving the protein structure of Artemis and further define its role in development of a healthy immune system.